Towards a membrane proteome in Drosophila: A method for the isolation of plasma membrane

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Abstract

Background: The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.Results: We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.Conclusion: A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.

Original languageEnglish (US)
Article number302
JournalBMC genomics
Volume11
Issue number1
DOIs
StatePublished - May 12 2010

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Proteome
Drosophila
Cell Membrane
Membranes
Density Gradient Centrifugation
Invertebrates
Dextrans
Lectins
Membrane Proteins
Genetic Models
Mitochondrial Membranes
Membrane Lipids
Microsomes
Drosophila melanogaster
Glycosylation
Diptera
Endoplasmic Reticulum
Proteomics
Vertebrates
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Genetics

Cite this

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title = "Towards a membrane proteome in Drosophila: A method for the isolation of plasma membrane",
abstract = "Background: The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.Results: We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37{\%} are integral membrane proteins from all compartments. Of the 432 proteins, 22{\%} have been previously assigned to the PM compartment, and a further 34{\%} are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.Conclusion: A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.",
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Towards a membrane proteome in Drosophila : A method for the isolation of plasma membrane. / Khanna, Mansi R.; Stanley, Bruce A.; Thomas, Graham H.

In: BMC genomics, Vol. 11, No. 1, 302, 12.05.2010.

Research output: Contribution to journalArticle

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